DNA methylation consisting of a cytosine modified by a methyl group at the N5 position (5meC) is a well described modification affecting gene expression in mammalian cells. The 5meC modification generally occurs at the CpG dinucleotide sequence; however, it has been identified elsewhere in the genome (Illingworth et al. (2009) FEBS Letters 583:1713-1720; herein incorporated by reference in its entirety). Another type of gene-regulatory DNA modification has more recently been described, 5-hydroxymethylcytosine (5hmC) (Tahiliani et al. (2009) Science 324:930-935; Kriaucionis et al. (2009) Science 324:929-930; each herein incorporated by reference in its entirety). Tahiliani et al. demonstrated that the enzyme Tet1, an iron-dependent α-ketogluterate dioxygenase, catalyzes the formation of 5hmC from 5meC (Tahiliani et al. (2009) Science 324:930-935; herein incorporated by reference in its entirety). Furthermore, the 5hmC base may be an intermediate in the conversion of 5meC to cytosine, thus identifying an enzyme that can potentially demethylate DNA (Tahiliani et al. (2009) Science 324:930-935; herein incorporated by reference in its entirety). 5hmC is a stable DNA modification found in specialized nondividing neurons and in all animal tissues studied to date (Kriaucionis et al. (2009) Science 324:929-930; herein incorporated by reference in its entirety). Intriguingly, 5hmC was not detected in cancerous cell lines. The inability to detect 5hmC in cancerous cell lines indicates that lack of 5hmC may be associated with tumorigenesis. The involvement of 5hmC in epigenetic regulation has been experimentally verified by Jin et al., who showed that 5hmC in DNA inhibits binding of several methyl-CpG-binding domain proteins (Jin et al. (2010) Nucleic Acids Res. 38:e125; Hendrich et al. (1998) Mol. Cellular Biol. 18:6538-6547; each herein incorporated by reference in its entirety). Tet2 and Tet3 also catalyze the formation of 5hmC (Ito et al. (2010) Nature 466:1129-1133; herein incorporated by reference in its entirety). Additionally, the total level of 5hmC present in several mammalian tissues has been quantified (Szwagierczak et al. (2010) Nucleic Acids Res. (advanced online publication); Munzel et al. (2010) Angewandte Chemie Intl. Ed. 49:5375-5377; each herein incorporated by reference in its entirety). However, specific genomic locations of 5hmC remain unknown.
The identification of specific genomic regions containing 5hmC is technically challenging. The most frequently used method for identifying 5meC, bisulfite sequencing, cannot distinguish 5meC from 5hmC (Nestor et al. (2010) Biotechniques 48:317-319; herein incorporated by reference in its entirety). Furthermore, commercially available antibodies raised against 5hmC cannot distinguish between 5meC and 5hmC (Ito et al. (2010) Nature 466:1129-1133; herein incorporated by reference in its entirety). Using polymerase kinetics, one group has been able to differentiate between 5meC and 5hmC (Flusberg et al. (2010) Nature Methods 7:461-465; herein incorporated by reference in its entirety). However, this method is impractical for identification of the 5hmC status of individual genes (Flusberg et al. (2010) Nature Methods 7:461-465; herein incorporated by reference in its entirety).
Improved methods for detecting 5-hydroxymethylcytosine residues in DNA are needed. In particular, improved methods for identifying the level of 5hmC modification at specific regions (e.g., specific gene loci) are needed.